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Modulatory effects of egg white ovotransferrin-derived tripeptide IRW (IleArg-Trp) in vascular smooth muscle cells against angiotensin II stimulation Wang Liao, Subhadeep Chakrabarti, Sandra T. Davidge, and Jianping Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03513 • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 21, 2016
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Journal of Agricultural and Food Chemistry
Modulatory effects of egg white ovotransferrin-derived tripeptide IRW (Ile-Arg-Trp)
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on vascular smooth muscle cells against angiotensin II stimulation
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Wang Liao, † Subhadeep Chakrabarti, †,∥,⊥ Sandra T. Davidge, ‡,§,∥,⊥ Jianping Wu *,†,∥
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†
Department of Agricultural, Food & Nutritional Science, ‡ Department of Obstetrics &
Gynecology,
§
Department of Physiology,
∥
Cardiovascular Research Centre, ⊥ Women and
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Children’s Health Research Institute
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University of Alberta, Edmonton, Alberta, T6G 2P5, Canada
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Corresponding author
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*E-mail:
[email protected]. 4-10 Ag/For Centre, University of Alberta, Edmonton, AB T6G
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2P5, Canada. Phone: (780) 492-6885. Fax: (780) 492-4265.
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ABSTRACT
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The renin angiotensin system (RAS) is a key mediator of blood pressure regulation. Angiotensin
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II (Ang II), the active component of RAS, is a potent vasoconstrictor that also causes abnormal
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proliferation, oxidative stress and inflammation in vascular smooth muscle cells (VSMCs) that
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contribute to atherosclerotic changes. Egg white ovotransferrin-derived tripeptide IRW (Ile-Arg-
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Trp) was previously shown to exert antihypertensive effect by reducing Ang II synthesis as well
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as endothelial cell inflammation and endothelial dysfunction. However, the effects of IRW on
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VSMCs are still unclear. In the present study, we evaluated the anti-proliferative, anti-oxidant,
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and anti-inflammatory effects of IRW on VSMCs in the presence of Ang II stimulation. It was
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found that IRW treatment could attenuate Ang II-stimulated proliferation, superoxide production
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and inflammation in VSMCs. These beneficial effects appeared to involve modulation of the NF-
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κB pathway. These findings could further our understanding on the antihypertensive mechanism
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of IRW beyond vascular endothelium.
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KEYWORDS: IRW, VSMC, proliferation, oxidative stress, inflammation
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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Hypertension is a global health challenge, accounting for about 6% of the total deaths
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worldwide.1 Hypertension is also a major risk factor for renal and cardiovascular diseases.2 The
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renin angiotensin system (RAS) is one of the major regulators of blood pressure and fluid and
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electrolyte homeostasis,3 in which, angiotensin II (Ang II) is the main effector peptide of
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vasoconstriction.4 Hence, angiotensin converting enzyme (ACE) inhibitor (to reduce the
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production of Ang II), or angiotensin II type I receptor (AT1R) blocker (to inhibit the binding of
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Ang II to its target tissues) are the first-line drugs for hypertension therapy.5
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Ang II is more than a potent vasoconstrictor. It also plays multiple roles in controlling the
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functional and structural integrity of the vascular wall.6 In vascular smooth muscle cells
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(VSMCs), over stimulation of Ang II could lead to aberrant proliferation,7 excessive superoxide
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production8 and inflammation,6 which would further promote vascular remodelling and
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inflammation, playing pivotal roles in atherosclerotic diseases.4 The anti-proliferative, anti-
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oxidant and anti-inflammatory actions of ACE-inhibitory agents have been documented.
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However, as pharmacological drugs require long-term (often life long) therapy with considerable
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side-effects, there is an unmet need for safer alternatives from naturally based sources.12,13
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In the past three decades, food protein-derived antihypertensive peptides have received
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substantial interest as natural alternatives to clinical drugs.14 Antihypertensive peptides derived
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from egg white, milk and soy proteins have been reported and the in vivo effects of some of these
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peptides have been validated on spontaneously hypertensive rats (SHRs).15-19 Functional food
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and nutraceuticals developed from these peptides may provide new opportunities for treatment
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and prevention of hypertension. IRW (Ile-Arg-Trp), an egg white ovotransferrin-derived
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tripeptide, was identified and characterized as a potent ACE inhibitor.20 The anti-hypertensive
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activity of IRW is associated with amelioration of endothelial inflammation and oxidative
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stress,21 enhancement of nitric oxide-mediated vasodilation17 and upregulation of angiotensin
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converting enzyme 2 (ACE2) expression.22 However, beneficial effects of IRW on VSMCs, if
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any, have not been investigated previously.
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Given this background, we aimed to investigate effects of IRW on proliferation, oxidative
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stress and inflammation in VSMCs under Ang II stimulation in this study.
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MATERIALS and METHODS
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Reagents. Dulbecco’s phosphate buffered saline (PBS), Ang II and dithiothreitol (DTT) were
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from Sigma Aldrich (St Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM) and
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fetal bovine serum (FBS) were purchased from Gibco/ Invitrogen (Carlsbad, CA, USA). Type 1
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Collagenase used for cell splitting was from Worthington Biochemical Corporation (Lakewood,
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NJ, USA). Penicillin-Streptomycin as well as Gentamicin was from Life Technologies (Carlsbad,
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CA, USA). Triton-X-100 was from VWR International (West Chester, PA, USA). The tripeptide
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IRW was synthesized by Genscript (Piscataway, NJ, USA). Peptide sequence and purity (99.8
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%) were validated by HPLC-MS/MS. The peptide was dissolved in PBS at a stock concentration
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of 10 mM, aliquoted and stored at -20οC until use.
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Cell culture. Rat aortic VSMC cell line A7r5 was purchased from ATCC (cat# ATCC CRL-
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1444, Manassas, VA, USA). The cells were grown in DMEM supplemented with 10% FBS and
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antibiotics (Penicillin-Streptomycin and Gentamicin) until they reached 80% confluence. For
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actual experiments, the confluent cells were placed in a quiescing medium (DMEM + 1% FBS +
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antibiotics) and then treated with 50 µM of IRW 1 h prior to the addition of 1 µM of Ang II for
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different time periods. Cells between passages 4 and 11 (since receiving) were used for all
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experiments.
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Cell proliferation assay. Cell proliferation was evaluated by bromodeoxyuridine (BrDU)
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incorporation assay. A7r5 cells were seeded into a 48 well plate and grown in DMEM with 10%
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of FBS for overnight. After the medium was replaced by quiescing medium, 50 µM of IRW was
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added 1 h prior to stimulation of Ang II (1 µM). The co-treatment period with IRW and Ang II
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was 23 h. Afterwards, BrDU reagent (Invitrogen) diluted in quiescing medium at a final
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concentration of 1% was added for 1 h incubation at 37 ℃. Then the cells were fixed by 70%
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ethanol (20 min, room temperature). The
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hydrochloric acid (20 min, room temperature) followed by permeabilization by 0.1% Triton-X-
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100 in PBS (5 min, room temperature) and finally, blocking was done in 1% bovine serum
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albumin (BSA) in PBS (60 min, room temperature). Mouse monoclonal antibody against BrDU
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(1:1000; Cell Signaling, Beverly, MA, USA) was diluted in PBS with 0.1% BSA and incubated
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at 4 °C overnight. Upon washing, cell was treated with anti-mouse secondary antibody
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(Molecular Probes, Eugene, OR, USA) for 30 minutes in the dark. Nuclei were stained with the
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Hoechst33342 nuclear dye (Molecular Probes). Visualization was performed by Olympus IX81
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fluorescent microscope (Carson Scientific Imaging Group; Markham, ON, Canada). For each
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data point, 3 fields were randomly selected and the number of nuclei counted. The percentage of
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nuclei positive for BrDU stain was noted in each field and the mean value was calculated.
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antigen was exposed by incubation with 1M
Superoxide detection. Cellular superoxide generation was detected by dihydroethidium (DHE)
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staining similar to our previous work.21 Reactive oxygen species (ROS) could react with DHE to
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form ethidium, which then binds to nuclear DNA and release nuclear fluorescence.23 A7r5 cells
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were seeded into 48 well plate and grown in DMEM with 10% of FBS until confluent. After the
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medium was replaced by quiescing medium, 50 µM of IRW was added 1 h prior to stimulation
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of Ang II (1 µM). The co-treatment period of IRW and Ang II was 30 min. Then cells were
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treated with 20 µM of DHE and incubated in dark for 30 min. Afterwards, the cells were washed
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3 times. Fluorescence signal was detected by Olympus IX81 fluorescent microscope (Carson
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Scientific Imaging Group). For each data point, images from 3 random fields were taken. Mean
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fluorescence intensity (MFI)
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(http://imagej.net/Welcome). MFI/cell was calculated based on the cell number in each field.
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Results were presented as % of the untreated group.
of
each
image was
quantified
by ImageJ
software
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Western blot. At the end of experiment, the A7r5 cells were lysed in boiling hot Laemmle’s
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buffer containing 50 mM DTT (a reducing agent) and 0.2% Triton-X-100 to prepare samples for
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western blotting. These cell lysates were then run in 9% sodium dodecyl sulfate polyacrylamide
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gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes and immunoblotted
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with antibodies. Protein bands for cyclooxygenase 2 (COX2) (rabbit monoclonal antibody from
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Abcam, cat# ab52237), inducible nitric oxide synthase (iNOS) (mouse monoclonal antibody
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from BD Transduction Laboratories, cat# 610432) and inhibitory κBα (IκBα) (Rabbit polyclonal
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antibody from Cell Signaling, cat# 9242) and AT1R (rabbit polyclonal antibody from Santa Cruz
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Biotechnology, cat# sc-1173) were normalized to the loading control α-tubulin (rabbit polyclonal
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antibody from Abcam, cat# ab15246). Phospho-p65 (p-p65) (rabbit polyclonal antibody from
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Cell Signling, cat# 3033) were normalized to the total p65 (mouse monoclonal antibody from
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Santa Cruz Biotechnology, cat# sc-8008). Anti-tubulin was used at 0.4 µg/ml, while all other
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antibodies were used at 0.5-1 µg/ml. Goat anti-rabbit IRDye 680RD or Donkey anti-mouse
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800CW from Licor Biosciences (Lincoln, NE, USA) was used as the secondary antibody. Protein
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bands were detected by Licor Odyssey BioImager (Licor Biosciences) and quantified by
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densitometry using Image Studio Lite 5.2 (Licor Biosciences). Cell lysates from untreated cells
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were loaded on every gel and all data were expressed as % of the corresponding untreated.
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Statistics. All data have been presented as mean ± SEM (standard error of mean) of 4 to 6
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independent experiments. Data were analyzed by one way analysis of variance (ANOVA) with
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Tukey’s post-hoc test. The PRISM 5 statistical software (Graph Pad Software, San Diego, CA)
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was used for all analyses. P
